Wounds In Diabetic Mice Research Articles

Topical treatment of ointment containing ethanol extract of Archidendron pauciflorum fruit peel on the wound healing in streptozotocin-induced diabetic mice

Malini DM, Madihah, Kamilawati F, Ratningsih N, Alipin K, Iskandar J. 2017. Topical treatment of ointment containing ethanol extract of Archidendron pauciflorum fruit peel on the wound healing in streptozotocin-induced diabetic mice. Nusantara Bioscience 9: 306-311. Diabetic wounds lead to severe tissue damage and are difficult to cure. One alternative medicine known well by local Indonesian communities to treat diabetic wounds is the fruit peel of djengkol. This study aimed to evaluate the ointment containing ethanol extract of djengkol fruit peel to accelerate wound healing process in the skin of streptozotocin-induced diabetic mice. The method was experimental using completely randomized design with six treatments and four replications. Diabetes was induced by intraperitoneal injection of streptozotocin 180 mg/kg BW. Mice with blood glucose level ≥150 mg/dL were used for diabetic mice models. The incision wound created at dorsolateral region of shaven skin at ±1 cm2 using sterile scissors. The ointment containing extract was applied topically to the diabetic mice wounds at concentration of 5%, 10% and 15%, as well as Betadine® as the reference group. The ointment basis was applied to the wound of diabetic mice as a positive control and to the wound of non-diabetic mice as a negative control. The treatment was done twice a day for 14 days. The results showed that topical application of ointment containing ethanol extract of djengkol fruit peel at concentration of 10% gave significant recovery (p<0.05) on the wounded skin by the enhancement of re-epithelization and granulation tissue, as well as the increase of capillary number and collagen density which were higher than other treatments and comparable to negative control group. It was concluded that the topical application of ointment containing ethanol extract of djengkol fruit peel can support the healing of diabetic wounds.

Anti-pseudomonal and anti-endotoxic effects of surfactin-stabilized biogenic silver nanocubes ameliorated wound repair in streptozotocin-induced diabetic mice

Pseudomonas aeruginosa and its lipopolysaccharides play a key role in the pathogenesis of diabetic foot infection, for which, currently no effective therapeutic agents are available. Hence, newer forms of therapeutic agents are required for treating Pseudomonas aeruginosa infection. In this present study, nanocrystalline silver nanoparticles (AgNPs) were synthesized using culture filtrate of Brevibacillus brevis KN8(2) followed by an investigation of its in vivo anti-pseudomonal and anti-endotoxic properties. Biosynthesized AgNPs was predominantly cubical in shape with an average particle size of 15.40 nm as observed through field emission scanning electron microscopy (FESEM) and X-ray diffraction analysis. The LC-ESI-MS/MS analysis indicates the presence of surfactin in culture filtrate of B. brevis KN8(2). The MIC of surfactin-stabilized AgNPs against P. aeruginosa was 10 μg ml−1 and its wound repair activity was observed in P. aeruginosa-infected wounds of diabetic mice by measuring wound area closure, bacterial counts, mRNA expressions, and histopathology. Further, surfactin-stabilized AgNPs suppressed the transcription of LPS-triggered expression of the TNF-α in wounds that LPS-assisted extension of wound repair in diabetes mellitus conditions was circumvented quite well. Results gathered in this study established that surfactin-stabilized AgNPs could effectively offer to the novel treatment of Gram-negative bacilli infection in diabetic wounds.

Quercus infectoria inhibits Set7/NF-κB inflammatory pathway in macrophages exposed to a diabetic environment

Chronic inflammation plays a key role in the pathogenesis of myriad complications associated with diabetes and thus anti-inflammatory therapies may ameliorate these complications. Quercus infectoria (Qi) extract has been shown to downregulate inflammatory processes; however, the molecular mechanisms of this anti-inflammatory activity remain unclear. The hypothesis of our study was that Qi extract exerts its anti-inflammatory effect by downregulating the Set7/NF-κB pathway. Bone marrow-derived macrophages (BMM) were treated with high glucose plus palmitate medium (HG/Pa) to simulate the diabetic environment. Compared with control conditions, HG/Pa elevated expression Set7, expression and activity of NF-κB along with expression of several inflammatory cytokines. These changes were associated with increased levels of intracellular reactive oxygen species (ROS). Moreover, similar alterations were demonstrated in BMM derived from mice fed a high fat diet (HFD) compared to those from lean mice, suggesting that HFD-induced changes in BM progenitors persist throughout differentiation and culture. Importantly, Qi extract dose-dependently reduced Set7, p65 and inflammatory cytokine expression relative to vehicle controls in both HG/Pa-and HFD-treated BMM. Finally, macrophages/monocytes isolated from wounds of diabetic mice that were treated with Qi solution exhibited lower expression of the inflammatory cytokines, IL-1β and TNF-α, compared with vehicle treated wounds, demonstrating translation to the in vivo diabetic environment. Taken together, data from this study suggests that Qi downregulates diabetes-induced activity of the Set7/NF-kB pathway.

Stromal Cell-Derived Growth Factor-1 Alpha-Elastin Like Peptide Fusion Protein Promotes Cell Migration and Revascularization of Experimental Wounds in Diabetic Mice.

Objective: In previous work, we demonstrated the development of a novel fusion protein containing stromal cell-derived growth factor-1 alpha juxtaposed to an elastin-like peptide (SDF1-ELP), which has similar bioactivity, but is more stable in elastase than SDF1. Herein, we compare the ability of a single topical application of SDF1-ELP to that of SDF1 in healing 1 × 1 cm excisional wounds in diabetic mice. Approach: Human Leukemia-60 cells were used to demonstrate the chemotactic potential of SDF1-ELP versus SDF1 in vitro. Human umbilical vascular endothelial cells were used to demonstrate the angiogenic potential of SDF1-ELP versus SDF1 in vitro. The bioactivity of SDF1-ELP versus SDF1 after incubation in ex-vivo diabetic wound fluid was compared. The in-vivo effectiveness of SDF1-ELP versus SDF1 was compared in diabetic mice wound model by monitoring for the number of CD31+ cells in harvested wound tissues. Results: SDF1-ELP promotes the migration of cells and induces vascularization similar to SDF1 in vitro. SDF1-ELP is more stable in wound fluids compared to SDF1. In vivo, SDF1-ELP induced a higher number of vascular endothelial cells (CD31+ cells) compared to SDF1 and other controls, suggesting increased vascularization. Innovation: While growth factors have been shown to improve wound healing, this strategy is largely ineffective in chronic wounds. In this work, we show that SDF1-ELP is a promising agent for the treatment of chronic skin wounds. Conclusion: The superior in vivo performance and stability of SDF1-ELP makes it a promising agent for the treatment of chronic skin wounds.

Blocking AGE-RAGE Signaling Improved Functional Disorders of Macrophages in Diabetic Wound.

Advanced glycosylation end products (AGEs) accumulate in diabetic wounds. Interactions between AGEs and their receptor (RAGE) leads to dermatologic problems in diabetes. Macrophage, which plays important roles in wound healing, highly expresses RAGE. Therefore, we investigated whether RAGE-expressing macrophages might be responsible for impaired wound healing on diabetes. We used anti-RAGE antibody applied topically on diabetic wounds. After confirming that wound healing was improved in anti-RAGE antibody group compared with normal mice, our results showed that macrophages appeared insufficient in the early stage and fading away slowly in the later proliferative phase compared with the control group, which was ameliorated in anti-RAGE antibody-applied wounds. Blocking AGE-RAGE signaling also increased neutrophils phagocytized by macrophages and promoted the phenotypic switch of macrophages from proinflammatory to prohealing activities. In vitro, phagocytosis of THP-1 (M0) and lipopolysaccharide- (LPS-) induced (M1) macrophages was impaired by treatment with AGEs, while IL-4- and IL-13-induced (M2) macrophages was not. Finally, AGEs increased the proinflammatory response of M1 macrophages, while inhibiting the polarization and anti-inflammatory functions of M2 macrophages. In conclusion, inhibition of AGE-RAGE signaling improved functional disorders of macrophages in the early inflammatory phase, which promoted the healing of wounds in diabetic mice.

A Small Chimeric Fibronectin Fragment Accelerates Dermal Wound Repair in Diabetic Mice.

Objective: During wound repair, soluble fibronectin is converted into biologically active, insoluble fibrils via a cell-mediated process. This fibrillar, extracellular matrix (ECM) form of fibronectin stimulates cell processes critical to tissue repair. Nonhealing wounds show reduced levels of ECM fibronectin fibrils. The objective of this study was to produce a small, recombinant wound supplement with the biological activity of insoluble fibronectin fibrils. Approach: A chimeric fibronectin fragment was produced by inserting the integrin-binding Arg-Gly-Asp (RGD) loop from the tenth type III repeat of fibronectin (FNIII10) into the analogous site within the heparin-binding, bioactive fragment of the first type III repeat (FNIII1H). FNIII1HRGD was tested for its ability to support cell functions necessary for wound healing, and then evaluated for its capacity to accelerate healing of full-thickness dermal wounds in diabetic mice. Results:In vitro, FNIII1HRGD supported cell adhesion, proliferation, and ECM fibronectin deposition. Application of FNIII1HRGD to dermal wounds of diabetic mice significantly enhanced wound closure compared with controls (73.9% ±4.1% vs. 58.1% ±4.7% closure on day 9, respectively), and significantly increased granulation tissue thickness (2.88 ± 0.75-fold increase over controls on day 14). Innovation: Recombinant proteins designed to functionally mimic the ECM form of fibronectin provide a novel therapeutic approach to circumvent diminished fibronectin fibril formation by delivering ECM fibronectin signals in a soluble form to chronic wounds. Conclusion: A small, chimeric fibronectin protein was developed. FNIII1HRGD demonstrated enhanced bioactivity in vitro and stimulated wound repair in a murine model of chronic wounds.

Polycationic Nanofibers for Nucleic Acid Scavenging.

Dying cells release nucleic acids (NA) and NA-containing complexes that activate inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation frequently encountered in autoimmune and inflammatory diseases. In this study, grafting of cationic polymers onto a nanofibrous mesh enabled local scavenging of negatively charged pro-inflammatory molecules in the extracellular space. Nucleic acid scavenging nanofibers (NASFs) formed from poly(styrene-alt-maleic anhydride) conjugated with 1.8 kDa bPEI resulted in nanofibers of diameters 486 ± 9 nm. NASFs inhibited the NF-κB response stimulated by the negatively charged agonists, CpG and poly(I:C), in Ramos-blue cells but not Pam3CSK4, a nonanionic agonist. Moreover, NASFs significantly impeded NF-κB activation in cells stimulated with damage-associated molecular pattern molecules (DAMPs) released from doxorubicin killed cancer cells. In vivo application of NASFs to open wounds demonstrated nucleic acid scavenging in wounds of diabetic mice infected with Pseudomonas aeruginosa, suggesting the in vivo efficacy of NASFs. This simple technique of generating NASF results in effective localized anti-inflammation in vitro and local nucleic acid scavenging in vivo.

Topical administration of cryopreserved living micronized amnion accelerates wound healing in diabetic mice by modulating local microenvironment.

Approximately 25% of diabetic patients suffer from diabetic lower-extremity ulcer throughout their lives and 7%-20% of patients will eventually need an amputation despite standard care treatment. The development of new therapies to treat diabetic wounds is urgent. In this study, we used cryopreserved living micronized amnion (300-600μm) to treat wounds in diabetic mice. Post-thaw micronized amnion retained high cell viability, as well as intact cell morphology and membrane structure. When transplanted onto the wounds of db/db mice, the cryopreserved living micronized amnion greatly promoted wound healing in diabetic mice mainly by secreting growth, inflammation, and chemotaxis-related factors that regulated macrophage migration and phenotype switch, recruited CD34+ progenitor cells, and increased neovascularization. In addition, the micronized amnion matrix can exist in the dermis and serve as a long-term dermal scaffold. These results demonstrated the potential of the cryopreserved living micronized amnion as a ready-to-use living dermal substitute that addresses multiple defective physiological processes of impaired wounds to treat diabetic ulcers and other chronic wounds in clinics.

Role of the autologous mesenchymal stem cells compared with platelet rich plasma on cicatrization of cutaneous wounds in diabetic mice

Abstract: Chronic cutaneous lesions affect 15% of diabetic human patients and represent a risk 15 to 46 times larger of limb amputations compared to people with normal glycemia. It is assumed that half of these amputations could be prevented by early treatment of wounds, for example, with proper cell therapy. Objectives: In this study, the action of the autologous transplant of mesenchymal stem-cells (MSC) was evaluated compared to the treatment with autologous platelet rich plasma (PRP) in the cicatrization of cutaneous lesions induced in diabetic mice. These animals were previously treated with streptozootocin to induce diabetes mellitus and round wounds of 1.5cm in diameter were created in the posterior region. Diameters of the wounds and healing time were evaluated during 30 days and the results were submitted to variance analysis and Tukey's test average. It was noticed that the animals treated with MSC presented a more accelerated cicatrization of the cutaneous lesion than the animals treated with PRP. However, the treatment with PRP presented better results than just the daily asepsis of the lesions with saline or covering them with semi-permeable bandage. Besides, the use of semi-permeable bandage kept the cutaneous lesions of diabetic mice did not interfere negatively with cicatrization, proved to be harmless to use, but kept the cutaneous lesions more hydrated than the ones exposed to the environment.

Protocol to Create Chronic Wounds in Diabetic Mice

Protocol to Create Chronic Wounds in Diabetic Mice

The development and characterization of SDF1α-elastin-like-peptide nanoparticles for wound healing

Chronic skin wounds are characterized by poor re-epithelialization, angiogenesis and granulation. Previous work has demonstrated that topical stromal cell-derived growth factor-1 (SDF1) promotes neovascularization, resulting in faster re-epithelialization of skin wounds in diabetic mice. However, the clinical usefulness of such bioactive peptides is limited because they are rapidly degraded in the wound environment due to high levels of proteases. Here, we describe the development of a recombinant fusion protein comprised of SDF1 and an elastin-like peptide that confers the ability to self-assemble into nanoparticles. The fusion protein and recombinant human SDF1 showed similar binding characteristics, as indicated by the measured equilibrium dissociation constant (Kd) for the binding of free SDF1 or the fusion protein to the CXCR4 receptor. The biological activity of SDF1-ELP, as measured by intracellular calcium release in HL60 cells was dose dependent, and also very similar to that of free SDF1. In contrast, the biological activity of SDF1-ELP in vivo was significantly superior to that of free SDF1. When applied to full thickness skin wounds in diabetic mice, wounds treated with SDF1-ELP nanoparticles were 95% closed by day 21, and fully closed by day 28, while wounds treated with free SDF1, ELP alone, or vehicle were only 80% closed by day 21, and took 42days to fully close. In addition, the SDF1-ELP nanoparticles significantly increased the epidermal and dermal layer of the healed wound, as compared to the other groups. These results indicate that SDF1-ELP fusion protein nanoparticles are promising agents for the treatment of chronic skin wounds.

Development of Stabilized Growth Factor-Loaded Hyaluronate- Collagen Dressing (HCD) matrix for impaired wound healing.

BackgroundDiabetes mellitus is a disease lack of insulin, which has severely delayed and impaired wound healing capacity. In the previous studies, various types of scaffolds and growth factors were used in impaired wound healing. However, there were several limitations to use them such as short half-life of growth factors in vivo and inadequate experimental conditions of wound-dressing material. Thus, our study aimed to determine the biocompatibility and stability of the matrix containing structurally stabilized epidermal growth factor (S-EGF) and basic fibroblast growth factor (S-bFGF).Results and DiscussionWe stabilized EGF and bFGF that are structurally more stable than existing EGF and bFGF. We developed biocompatible matrix using S-EGF, S-bFGF, and hyaluronate– collagen dressing (HCD) matrix. The developed matrix, S-EGF and S-bFGF loaded on HCD matrix, had no cytotoxicity, in vitro. Also, these matrixes had longer releasing period that result in enhancement of half-life. Finally, when these matrixes were applied on the wound of diabetic mice, there were no inflammatory responses, in vivo. Thus, our results demonstrate that these matrixes are biologically safe and biocompatible as wound-dressing material.ConclusionsOur stabilized EGF and bFGF was more stable than existing EGF and bFGF and the HCD matrix had the capacity to efficiently deliver growth factors. Thus, the S-EGF and S-bFGF loaded on HCD matrix had improved stability. Therefore, these matrixes may be suitable for impaired wound healing, resulting in application of clinical treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s40824-016-0056-4) contains supplementary material, which is available to authorized users.

Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds.

Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms and by the responding leukocytes, may impede wound healing by depleting the oxygen that is required for healing. In this study, oxygen microsensors to measure oxygen transects through in vitro cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse wound model, and ex vivo human chronic wound specimens was used. The results showed that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17 to 72 mmHg on live mice and from 6.4 to 1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. To characterize the metabolic activities of the bacteria in the mouse scabs, transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds was performed. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results also indicated that the bacteria within the wounds experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results supported the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions, through their metabolic activities and through their recruitment of cells that consume oxygen for host defensive processes.

Additive enhancement of wound healing in diabetic mice by low level light and topical CoQ10.

Diabetes, a highly prevalent disease that affects 9.3% of Americans, often leads to severe complications and slow wound healing. Preclinical studies have suggested that low level light therapy (LLLT) can accelerate wound healing in diabetic subjects, but significant improvements must be made to overcome the absence of persuasive evidence for its clinical use. We demonstrate here that LLLT can be combined with topical Coenzyme Q10 (CoQ10) to heal wounds in diabetic mice significantly faster than LLLT alone, CoQ10 alone, or controls. LLLT followed by topical CoQ10 enhanced wound healing by 68~103% in diabetic mice in the first week and more than 24% in the second week compared with untreated controls. All wounds were fully healed in two weeks following the dual treatment, in contrast to only 50% wounds or a fewer being fully healed for single or sham treatment. The accelerated healing was corroborated by at least 50% higher hydroxyproline levels, and tripling cell proliferation rates in LLLT and CoQ10 treated wounds over controls. The beneficial effects on wound healing were probably attributed to additive enhancement of ATP production by LLLT and CoQ10 treatment. The combination of LLLT and topical CoQ10 is safe and convenient, and merits further clinical study.

Regulation of impaired angiogenesis in diabetic dermal wound healing by microRNA-26a

Wound healing is a physiological reparative response to injury and a well-orchestrated process that involves hemostasis, cellular migration, proliferation, angiogenesis, extracellular matrix deposition, and wound contraction and re-epithelialization. However, patients with type 2 diabetes mellitus (T2D) are frequently afflicted with impaired wound healing that progresses into chronic wounds or diabetic ulcers, and may lead to complications including limb amputation. Herein, we investigate the potential role of microRNA-26a (miR-26a) in a diabetic model of wound healing. Expression of miR-26a is rapidly induced in response to high glucose in endothelial cells (ECs). Punch skin biopsy wounding of db/db mice revealed increased expression of miR-26a (~3.5-fold) four days post-wounding compared to that of WT mice. Local administration of a miR-26a inhibitor, LNA-anti-miR-26a, induced angiogenesis (up to ~80%), increased granulation tissue thickness (by 2.5-fold) and accelerated wound closure (53% after nine days) compared to scrambled anti-miR controls in db/db mice. These effects were independent of altered M1/M2 macrophage ratios. Mechanistically, inhibition of miR-26a increased its target gene SMAD1 in ECs nine days post-wounding of diabetic mice. In addition, high glucose reduced activity of the SMAD1-3′-UTR. Diabetic dermal wounds treated with LNA-anti-miR-26a had increased expression of ID1, a downstream modulator or SMAD1, and decreased expression of the cell cycle inhibitor p27. These findings establish miR-26a as an important regulator on the progression of skin wounds of diabetic mice by specifically regulating the angiogenic response after injury, and demonstrate that neutralization of miR-26a may serve as a novel approach for therapy.

Bifunctional Therapeutic High-Valence Silver-Pyridoxine Nanoparticles with Proliferative and Antibacterial Wound-Healing Activities.

The antibacterial and moisturizing effects inherent to silver nanoparticles contribute greatly to their use as a topical antibacterial agent. The antibacterial property of silver nanoparticles provides topical wounds with an indirect environment for healing through the prevention of pathogenic infection. However, the direct wound-healing effects of silver nanoparticles have not been previously explored. In this work, we report a bimodal therapeutic silver nanoparticle that possesses both direct wound-healing and antibacterial properties. The nanoparticles consist of high-valence silver-pyridoxine complexes. The wound-healing efficacy was verified in diabetic mice, as well as in vitro assays. A MAPK pathway study demonstrated that silver-pyridoxine nanoparticles induced the proliferation and migration of keratinocyte and fibroblast cells. Antibacterial activities in 8 different pathogenic bacteria responsible for the infection of burn wounds were tested. The rapid wound healing occurring on skin wounds of diabetic mice attests to the utility of bimodal therapeutic silver nanoparticles as a next-generation topical therapeutic agent.

Loading and release of porous silicon nanoparticles with Flightless I neutralizing antibodies to aid wound healing

Event Abstract Back to Event Loading and release of porous silicon nanoparticles with Flightless I neutralizing antibodies to aid wound healing Steven Mcinnes1*, Christopher T. Turner1*, Allison J. Cowin1* and Nicolas H. Voelcker1* 1 University of South Australia,, Mawson Institute, Australia Introduction: Flightless I (Flii) is upregulated in acute and chronic wounds and is a negative regulator of wound healing[1]. Flii neutralizing antibodies (FnAbs) are able to improve the rate of wound healing and decrease scarring[2]. However, retaining FnAb activity in a hostile environment, such as a diabetic wound, is challenging. The aim of this work is to incorporate FnAb into biocompatible and biodegradable porous silicon[3] nanoparticles (pSiNPs) to protect them from the proteolytic degradation in chronic wounds. We investigated the ability of the pSiNP materials to effectively deliver FnAb to diabetic mouse wounds. Materials and Methods: A pSi membrane was fabricated by anodization of p-type Si wafers in an 18 cm2 etching cell in 3:1 HF:ethanol (v/v) solution using a square waveform (50 mA/cm2 for 7.3 s and 400 mA/cm2 for 0.3 s, 474 repeats) followed by electropolishing in 1:20 HF:ethanol at 4 mA/cm2 for 250 s. The freestanding pSi membrane was fractured into porous silicon nanoparticles (pSiNPs) by ultra-sonication for 16 h in DMSO. The resulting NPs solution was spun at 10,000 × g then filtered through a 220 nm PTFE syrine filter before centrifugation at 22000 × g. The pSiNPs were loaded with FnAb by incubating in 1 mg/mL solutions in PBS for 1 hour at 4 oC. UV-Vis based loading quantification was performed on the supernatant, after centrifugation at 16000 x g for 1 minute. Results and Discussion: pSiNPs loaded with FnAb, typically at loadings of up to 724.39 ± 34.79 μg of FnAb per mg of pSi, were incubated with keratinocytes. We observed an increase in cell proliferation and wound closure similar to that seen with FnAb alone, confirming that the released FnAb had not undergone any structural alteration or lost functionality (Fig 1A). To confirm a protective effect, IgG loaded pSiNP were incubated with proteases, rinsed, and then allowed to release the IgG at 25 oC for 7 days. The supernatant was collected and showed intact IgG for up to 7 days post-treatment, suggesting the pSiNPs were protecting the antibodies from the proteolytic environment (Fig 1B). Using a streptozotocin-induced murine model of diabetic wound healing, the effect of the FnAb-pSiNPs was assessed on healing. FnAb loaded pSiNPs were intradermally injected into excisional wounds at day 0 with healing assessed at day 7. A significant improvement in healing was observed compared to controls. Additionally, healing was more effective with FnAb-pSiNPs than with FnAb alone (Fig 2). Figure 1: A) Electric cell impedance sensing. Confluent HaCaTs were treated with FnAb-NPs (t = 0), and wounded (2,500 µA, 48,000 Hz) for 30 seconds (t = 1.5 hrs). Cells recovery was then monitored (24,000 Hz) for 10 hrs. Cells were treated with FnAb-NPs (25 ug/well NP; red, 12.5 ug/well NP; blue) and unloaded NPs (black). n = 3, with data presented as mean +/- 1 SD. B) Effect of proteases on IgG-loaded pSi. IgG loaded pSi was incubated with proteases, washed and then incubated for 7 days at 25 oC. Daily supernatants were run on coomassie-stained SDS-PAGE gels. Figure 2: Excisional wound trial. 6mm excisional wounds were treated with FnAb-NPs intradermally injected at the wound margins. All treatments contained 50 ug of the FnAb or IgG control. Mice were humanely killed at day 7, with wounds examined macroscopically (a, b). Data in (a) is wound area at day 7 as % of original size. * P<0.05. ** P<0.005. FnAb-NP = FnAb loaded pSiNPs, FnAb + NP = Separate FnAb and pSiNPs injections. Conclusions: pSiNPs are able to protect antibody based payloads from a proteolytic environment as well as provide a novel and effective vehicle for delivering antibodies to wound environments. These materials may also be fabricated into bandages to help develop novel non-invasive therapeutic approaches for wound healing. This research was in part conducted and funded by the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (project number CE140100036).

Protease-sensitive atelocollagen hydrogels promote healing in a diabetic wound model.

The design of exudate-managing wound dressings is an established route to accelerated healing, although such design remains a challenge from material and manufacturing standpoints. Aiming towards the clinical translation of knowledge gained in vitro with highly-swollen rat tail collagen hydrogels, this study investigated the healing capability in a diabetic mouse wound model of telopeptide-free, protease-inhibiting collagen networks. 4-Vinylbenzylation and UV irradiation of type I atelocollagen (AC) led to hydrogel networks with chemical and macroscopic properties comparable to previous collagen analogues, attributable to similar lysine content and dichroic properties. After 4 days in vitro, hydrogels induced nearly 50 RFU% reduction in matrix metalloproteinase (MMP)-9 activity, whilst showing less than 20 wt% mass loss. After 20 days in vivo, dry networks promoted 99% closure of 10 × 10 mm full thickness wounds and accelerated neo-dermal tissue formation compared to Mepilex®. This collagen system can be equipped with multiple, customisable properties and functions key to personalised chronic wound care.

Self‐Assembled Wound Dressings Silence MMP‐9 and Improve Diabetic Wound Healing In Vivo

The direct local delivery of short interfering RNA (siRNA) into target tissues presents a real solution to several complex medical conditions that today lack efficacious therapies. The development of an ultrathin polymer coating is described to sustain the delivery of siRNA for up to 2 weeks in vitro and in vivo. This technology successfully reduces the expression of MMP-9 within the wounds of diabetic mice, significantly accelerating the wound healing process and improving the quality of tissue formed.